NH2-TERMINAL PROLINE ACTS AS A NUCLEOPHILE IN THE GLYCOSYLASE AP-LYASE REACTION CATALYZED BY ESCHERICHIA-COLI FORMAMIDOPYRIMIDINE-DNA GLYCOSYLASE (FPG) PROTEIN/

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent ro le in the repair of oxidatively damaged DNA in Escherichia coli, The p rotein possesses three enzymatic activities, hydrolysis of the N-glyco sidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-e limination; these functions act in a concerted manner to excise oxidiz ed deoxynucleosides from duplex DNA, Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA comple x can be trapped by reduction with sodium borohydride, By digesting th e stable, co-valently linked intermediate with proteases and determini ng the accurate mass of the products by negative electrospray ionizati on-mass spectrometry, we show that the N-terminal proline of Fpg prote in is linked to DNA and, therefore, is identified as the nucleophile t hat initiates the catalytic excision of oxidized bases from DNA, This experimental approach may be applicable to the analysis of other prote in-DNA complexes.